Toxins and activities described in
1. Introduction
Few species of butterflies and moths (order Lepidoptera) are involved in human envenoming [1]. Caterpillars are the larval forms of moths and butterflies. Toxins are usually found in the caterpillar’s hairs and spines with defense purposes. The majority of medically important encounters with lepidopterans occur with exposure to the caterpillar’s urticating hairs or spines, but hemolymph can also have toxic properties [1, 2]. A variety of clinical effects have been described, which depend on the family and species involved, ranging from local to systemic reactions [3, 4].
In most occasions, the adverse effects caused by caterpillars are self-limited and can be treated with topical antipruritics [4]. However, for the envenoming by the South American
In 1989, an outbreak of accidents with this species became a serious public health threat in southern Brazil, with high fatality rates [5, 11-15]. Since then, many studies have been carried out to understand the pathophysiological mechanisms of envenoming [14] and to identify the toxins responsible for adverse reactions.
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Prothrombin activation (Lopap) | Bristle extract | 21 | Serine protease, activity increased by Ca2+; consumption coagulopathy |
[18, 19, 21, 24-26] |
FXa-like | Bristle extract | 21 | Hydrolytic activity on S-2222 chromogenic substrate, Ca2+-independent; N-terminal sequence similar to Lopap. | [27] |
Factor X activation (Losac) | Bristle extract | 45 | Serine protease, Ca2+-independent; Cell survival in HUVEC. Recombinant form produced in bacteria. |
[20-22, 28] |
Phospholipase A2-like | Bristle extract | 15 | Indirect hemolytic activity in human and rat red blood cells |
[29-31] |
Fibrinogenolytic (Lonofibrase) | Hemolymph | 35 | αβ fibrinogenase activity; enable to affect fibrin cross-linked. | [32-34] |
Hyaluronidase (Lonoglyases) | Bristle extract | 49 53 |
β-endohexosaminidase activity; degradation of extracellular matrix. | [35] |
Antiapoptotic | Hemolymph | 51 | Activity on |
[36] |
Antiviral | Hemolymph | 20 | Antiviral activity against measles, influenza and polio viruses. Recombinant form produced in baculovirus/insect. | [37, 38] |
Nociceptive and Edematogenic | Bristle extract | NI | Nociception facilitated by prostaglandin production; edematogenic response facilitated by prostanoids and histamine. | [39] |
Kallikrein-kinin system activation | Bristle extract | NI | Kinin release from low molecular weight kininogen; edema formation and fall in arterial pressure. | [40] |
Platelet adhesion and aggregation | Bristle extract | NI | Direct platelet aggregation and ATP secretion; activity inhibited by |
[41, 42] |
The principal components in the caterpillar’s venom have been initially identified by isolating toxins through classical purification methods and following the main activities observed in the whole bristle extract (Figure 2). However, this approach provides knowledge restricted only to the most abundant toxins, and usually reveals that activities which are directly associated to the main symptoms and effects observed in the envenoming outcomes. Experimental assays were specifically developed to test the hemostatic and enzymatic activities of
In the last years, methods applied in genomic, transcriptomic and proteomic analyses have been applied with the aims of cataloging and classifying the toxins based on their structure and activity (Figure 3). Thus, it was possible to analyze the envenoming processes at the molecular level. For example, significant advance was achieved through two independent transcriptome studies, which generated a list of putative toxic proteins from
Production of recombinant forms of
Additional studies will be conducted to determine the involvement of the venom components in the envenoming syndrome and their biological significance for physiological processes of the animal, such as insect metamorphosis, which is a combination of growth/activation/differentiation/programmed cell death signals. Thus, this chapter reviews the currently available information about
2. Molecular approach
For many years, direct purification of toxins from venoms was the best procedure to characterize them with regard to their primary structure. Then, the development of molecular approaches to characterize toxin genes represented an expansion in the understanding of the structure and function of toxin, critical for the development of new treatments directed against the venom toxins (antivenoms). Cloning of cDNAs coding for biochemically isolated toxins has improved their characterization.
2.1. Transcriptomics of Lonomia obliqua bristle extract
Expressed Sequence Tags strategy is an approach to characterize the transcriptome of a cell, gland or organism and is based in all the transcript (the most abundant are the mRNAs) produced at a specific time and fully sequenced to create a representative catalogue of expressed genes [47]. Hundreds to a thousand of sequences are grouped into
EST-strategy was used to identify the major transcripts present in
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General Metabolism | 72 | 94 | 1.30 | 6.1 | 7.0 |
Transcriptional and translational | 165 | 462 | 2.80 | 30.7 | 37.0 |
Processing and sorting | 10 | 13 | 1.30 | 0.8 | 1.0 |
Degradation | 9 | 20 | 2.22 | 1.3 | 2.0 |
Structural functions | 47 | 243 | 5.17 | 16.2 | 19.0 |
Cell regulation | 26 | 82 | 3.15 | 5.4 | 6.0 |
Other functions | 138 | 244 | 1.77 | 16.2 | 19.0 |
Conserved unknown proteins | 42 | 120 | 2.86 | 8.0 | 9.0 |
TOTAL | 509 | 1278 | 2.51 | 85.0 | 100.0 |
Other cDNA libraries were constructed from bristles and integument [43]. The transcripts of those libraries revealed the presence of sequences related to trypsin-like enzymes, blood coagulation factors, prophenoloxidase cascade activators, cysteine proteases, phospholipase A2, serpins, cystatins, antibacterial proteins, lipocalins, and others (GenBank accession number: AY829732–AY829859) [43]. Sequences deposited independently in gene banks from both cDNA libraries are complementary. Apart from new venom component precursors, both libraries describe gene products related to cellular processes important for venom production, including high protein synthesis, tuned post-translational processing and trafficking. Those important projects contributed significantly to the characterization of this venom, which showed to be a rich source of proteins and active principles. Further studies about the biological and pharmacological properties of these molecules are necessary to understand its involvement in the envenoming process. Recently, the next-generation of sequencing methods - for example, pyrosequencing - have improved and increased the sequencing reducing time and cost compared to the traditional Sanger method [47, 56].
2.2. Microarray analysis
The identification of genes expressed in cells of a tissue is a basic step to provide essential information about gene function and tissue physiology. The gene expression analysis through the microarray technology (cDNA arrays) has become a powerful tool for rapid analysis of the functional effects of toxins on cells and tissues [57]. The main application of cDNA arrays is to compare the expression of known genes in different physiological situations, for example, tissues in normal and pathological conditions [58]. Thus, analyses of array data contribute to a better understanding of complex gene expression patterns related to physiology and metabolism, unveiling networks or pathways previously unknown.
A study of the effects of
2.3. Immunoproteome of Lonomia obliqua bristle extract
The identification of antigens eliciting an immune response by applying proteomic technologies can be defined as
In classical gel-based strategy, the isolation and identification of proteins/antigens comprises a combination of bidimensional electrophoresis, immunoblotting and mass spectrometry. The aim of bidimensional electrophoresis is to isolate proteins based on their charge and mass [60, 61]. The first step is isoelectric focusing (IEF), where proteins migrate to reach their isoelectric point in an immobilized pH gradient gel under high voltage. All proteins are given negative charge by addition of SDS detergent. This step also includes denaturation of proteins by reduction and alkylation. The second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), where smaller proteins migrate faster through the gel to the anode than larger ones. Detection of proteins can be performed by gel staining or immunoblotting.
Immunoblotting involves the transfer of proteins from gel to a nitrocellulose or PVDF membrane in an electric field [62]. The immobilized proteins in the membrane are subsequently incubated with antibodies that have affinity for the proteins of interest. Detection is carried out by enzyme-labelled secondary antibodies against the constant region of the primary IgG antibody, followed by the addition of a chemilluminescent substrate. The substrate reaction can be visualized by fluorescence.
Following this, the immunogenic proteins are removed from the gel and enzymatically digested for further mass spectrometry analyses [63]. Trypsin is generally used, cleaving an amide bond on the C-terminal side of lysine and arginine residues, which will be protonated and analyzed in positive-ion mode. Addition of diluted acid (0.1% formic acid or 1% trifluoracetic acid) to the sample contributes to the ionization process.
The ionization methods that are most often used for peptides and proteins are Matrix Assisted Laser Desorption Ionization (MALDI) and Electrospray Ionization (ESI). Peptides and proteins can be identified by Peptide mass fingerprinting (PMF) or
The identification of immunogenic compounds from
Besides the biochemical and pharmacological tests, the quality control of serum and vaccine production can be monitored by proteomic technologies [64], such as chromatographic analyses, bidimensional electrophoresis and immunoblotting, once they are able to detect protein degradation and also confirm the presence of specific antibodies. However, immunotherapy can be more effective if a better characterization of venom composition is performed, improving immunization procedures, increasing its specificity and reducing side effects. The new generation of high affinity antibodies against low abundant immunogenic toxins can be evaluated by an antivenomic approach [64, 65].
A novel approach is the investigation of post-translational modifications (PTM) that affect antigen recognition, given that many peptides presented to T cells by the major histocompability complex are post-translationally modified [66]. Glycosylation and phosphorylation are important PTMs of proteins, playing crucial roles in several biological processes, including cell recognition and signalling pathway [67, 68]. Some potential targets for cancer therapy are based on glycosylated and phosphorylated epitopes discoveries [59].
Otherwise, phosphorylated proteins are usually enriched by immunoprecipitation (mainly for phosphotyrosine peptides) or by chromatographic procedures, such as Strong Cation eXchange (SCX), Hydrophilic Interaction Liquid Chromatography (HILIC), Immobilized Metal Affinity Chromatography (IMAC) or Metal Oxide Affinity Chromatography (MOAC). Different metals may be used (iron, zirconium, gallium, etc) and peptides eluted by acidic or basic conditions, releasing mono-phosphorylated and multi-phosphorylated peptides, respectively [67].
The simultaneous screening of thousands of proteins from complex samples in a fast and sensitive manner can be performed using protein arrays. Amongst the different protein microarray applications are biomarker discovery, protein interaction studies, enzyme-substrate profiling, immunological profiling and vaccine development. As our interest is in the immune response, an antibody microarray can be used for identification of antigens that react specifically with the antibodies spotted on a solid support, with the complex formed then detected by fluorescence [59].
A large number of not yet identified proteins are considered as unknowns, but higher probabilities of identifications are reached when different methodologies are applied for analysis of complex samples. The combination of several proteomic techniques described here could improve the detection of immunogenic compounds and create new perspectives for effective immunotherapies.
3. Lonomia obliqua toxins
The
Several molecules and activities were reported in bristles or hemolymph (Table 1). Some of them are related to the pathophysiology of the envenoming others to the development process of the animal such as regulation of the cell cycle [16]. Donato and colleagues [70] identified in the bristle extract a direct factor X activator which is calcium-independent, and a prothrombin activator. The prothrombin and factor X activators were later isolated and named Lopap and Losac, respectively [22, 24]. Interestingly, both molecules are no longer similar with any well-known procoagulant molecule from human or any other species.
3.1. Lopap: Functional characterization, recombinant production and bioinformatics analysis
Lopap (
The recombinant protein (rLopap) was obtained in enzymatically active form as monomer of 21 kDa with a polyhistidine tag after purification by immobilized metal-chelate affinity chromatography. Partial amino acid sequences of native Lopap lead to identification of its respective clone from the cDNA library of
Interestingly, the deduced amino acid sequence of Lopap showed no similarity with other prothrombin activators or serine proteases, but was similar to lipocalin family members, either from insects or mammals [71]. Lopap sequence alignment with other lipocalins is shown in Figure 8. Members of lipocalin family usually share only about 30% of similarity in amino acid sequence, despite showing conserved secondary and tertiary structures. Furthermore, these proteins have in primary structure three characteristic conserved motifs [72].
Lopap’s tridimensional structure obtained by molecular modeling has the characteristic fold of lipocalins, consisting in an eight stranded antiparallel β-barrel (Figure 9) with a hydrophobic pocket for binding of hydrophobic ligands. A serine protease catalytic triad was also predicted [19]. Lopap is the first lipocalin described that displays proteolytic activity. On the other hand, through a peptide mapping approach based on lipocalin conserved motifs found in the Lopap’s primary structure, a synthetic peptide was obtained (Figure 10), which has been proposed as a sequence signature among lipocalins, sharing a common role in cell protection and development process [73]. Other lipocalins that have been described with antiapoptotic activity share similar sequences, which have similar conformations in their tridimensional structures [73].
Lopap shows specific proteolytic activity towards prothrombin. It displays serine protease-like activity and activates human prothrombin through hydrolysis of Arg284-Thr285 and Arg320-Ile321 peptide bounds, generating active thrombin, without formation of the intermediate meizothrombin [24]. This mechanism is similar to prothrombin activation by FXa in absence of the prothrombinase complex (Figure 11), previously described [74]. This is the unique prothrombin activation mechanism described for an exogenous serine protease, which is independent of prothrombinase complex components. All other exogenous prothrombin activators (metalloproteases and serine proteases) currently described from snake venoms fit into four groups, sharing similar mechanisms of action [75].
When administered
The Lopap-derived peptide obtained through chemical synthesis (Survicalin) reproduces the Lopap’s modulation on endothelial cells and neutrophils cell, triggering antiapoptotic activity [73]. Survicalin also induces fibroblast responses, decreasing caspase-3 and increasing Bcl-2, Ki-67, IL-1β and the receptors for IL-8 and IL-6. Enhanced production of extracellular matrix proteins, such as collagen, fibronectin, tenascin and laminin is also induced by Survicalin in fibroblast culture [79].
3.2. Losac: Functional characterization
Losac is the first factor X activator purified from a lepidopter secretion. It was obtained from caterpillar’s bristle extract as a single polypeptide chain protein of about 45 kDa [22]. Some years later, from a cDNA library of
Biochemical characterization of Losac has shown that, although its sequence did not show an equivalent among other factor X activators, Losac possess a similar mechanism of action than RVV-X, a factor X activator purified from Russell’s viper venom
A model proposed by Morita [83] and crystallographic studies of RVV-X [85] support the hypothesis that it primarily recognizes the calcium-bound conformation of Gla-domain in factor X through an exosite formed by the light chains, followed by the catalytic conversion of factor X to factor Xa. Despite the structural differences between Losac and RVV-X, it remains possible that they share a similar mechanisms for recognition of factor X involving calcium ions, phospholipids and the Gla-domain of factor X followed by its proteolytic conversion to active factor X.
Besides its role in coagulation [22, 28], Losac is also capable of inducing proliferation and inhibiting endothelial cell death while stimulating the release of NO, a known molecule with antiapoptotic activity [86, 87], and t-PA, a component of fibrinolytic-pathway involved in matrix remodeling [88]. The authors suggest that the cell proliferation and cell viability activities elicited by Losac are probably related to the NO liberation [22], since NO was also described as an endothelial survival factor, inhibiting apoptosis [86, 87]. Moreover, it was also observed that the production/expression of some important molecules involved in inflammation and coagulation systems such as ICAM-1, PGI2, DAF, IL-8, vWF and tissue factor were not affected by Losac.
It has been show that hemolymph from some insects can increase cell longevity by inhibiting apoptosis [89, 90]. The increase of
3.3. Molecular cloning and heterologous expression of Losac
The production of Losac in a recombinant form was important due to the disadvantages of purifying Losac from bristle extract: the use of many caterpillars to prepare the bristle extract and the low yield of native Losac (0.3%) [22]. Cloning and production scheme to obtain rLosac is shown in Figure 12 [28].
Nucleotide and deduced amino acid sequences were compared with data banks in order to identify similar genes and their products. The analysis revealed a high similarity with members of the immunoglobulin-like superfamily of cell adhesion molecules (IgCAMs), especially with neural CAMs (NCAMs) [28]. Members of this group have diverse functions but none was associated with proteolytic activities [94]. Multiple comparison of the deduced amino acid sequence revealed different degrees of identity with IgCAMs: 26% of identity with L1-NCAM from humans, 34% with the protein neuroglian from
At this stage we can only speculate about the mechanism of action of Losac. One possibility is to evaluate structural features that might contribute to the Losac-induced factor X activation. Thus, a search for serine protease active-site was undertaken based on the
Functionally, hemolins were first associated with the insect immune system because of their over-expression after bacterial infection [103]. Due to their adhesion properties, some hemolins have been involved in the cell adhesion mechanisms. In the last two decades independent studies demonstrated that hemolins are multifunctional molecules involved in a diverse range of cell interaction [104-110]. The high identity among Losac and hemolins suggests that Losac could also assume some of these functions in
4. Biomedical applications
Studies on
Modulation of cell responses triggered by
Unveiling the mechanisms of action and structure-function relationship of these multifunctional molecules may pointing out these molecules as promising candidates to development of new therapeutic drugs, reagents in diagnostic kits for coagulation dysfunctions, and biotechnological applications.
5. Concluding remarks
Nature has been finding ways to gift living beings with functions that are advantageous, regarding natural selection, mainly by evolutionary process. Among all the lepidopterans of medical interest in the world,
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WO/2003/070746 | 08.28.2003 | Purification and characterization of a prothrombin activator from the bristle of Lonomia obliqua: to be used in diagnosis kits for detecting plasma prothrombin in hemorrhagic state patients | Instituto Butantan (Brazil); Fapesp (Brazil) and Biolab Sanus Farmacêutica Ltda (Brazil) |
WO/2006/021062 | 02.03.2006 | Process for obtaining the recombinant prothrombin activating protease (rLopap) in monomeric form; the recombinant prothrombin activating protease (rLopap) as well as its amino acid sequence; the use of this protease as a defibrinogenase agent and the diagnosis kit for dysprothrombinemias | Instituto Butantan (Brazil); Fapesp (Brazil) and Biolab Sanus Farmacêutica Ltda (Brazil) |
WO/2007/028223 | 03.15.2007 | Lopap-based pharmaceutical compositions and uses thereof: it refers to the use of Lopap as modulators of cell death and degeneration caused by wounds, aging and external agents | Instituto Butantan (Brazil); Fapesp (Brazil) and Biolab Sanus Farmacêutica Ltda (Brazil) |
WO/2009093189 | 07.30.2009 | Peptides, compositions, and uses thereof: it refers to the uses of Lopap-derived peptides for regenerating tissues and wound repair | Instituto Butantan (Brazil); Fapesp (Brazil) and Biolab Sanus Farmacêutica Ltda (Brazil) |
Application of molecular approaches in the study of
Integrating transcriptomic, proteomic and microarray analysis will provide a wealth of valuable information about venom composition. Molecular cloning and expression of recombinant toxins from
Acknowledgments
The authors thank the Brazilian founding agencies FAPESP and CNPq, INCTTOX program. L.C.C.-C. had a Post-Doctoral fellowship from CAT-CEPID/FAPESP.
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